Identification of ribosomal protein S1 as a poly(A) binding protein in Escherichia coli.
To elucidate the metabolic function of mRNA polyadenylation in Escherichia coli. we searched for a polyadenylate-binding protein as a potential mediator of the function of the poly(A) moiety. Using a nitrocellulose filter-binding assay and a Northwestern blot technique, a protein in the ribosomal supernatant fraction of E coli was identified and purified to homogeneity. N-terminal sequence analysis yielded a 25-residue sequence which corresponded to the 25 N-terminal amino acids of protein S1, one of the proteins of the E coli 30S ribosomal subunit. Poly(A) binding to S1 protein was inhibited by Mg2+ and Mn2+ and by ATP and stimulated 8-fold by 100 mM KCl. The binding of S1 to poly(A) occurred with an association constant of 3 x 10(6) M-1 and seemed to be only mildly cooperative. Competition studies of the binding of poly(A) and poly(C) to purified S1 protein were consistent with the presence of two polynucleotide binding sites, of which one binds poly(A) five times more strongly than poly(C), whereas the other binds poly(C) 50 times more strongly than poly(A). Poly(A) bound to 30S ribosomal subunits but not to 50S ribosomes. To study possible association of S1 with the poly(A) tracts of E coli mRNA in the process of translation, poly(A) RNA was isolated from polysomes by oligo(dT) cellulose chromatography and the poly(A) RNA with bound protein was eluted either directly or after digestion with RNase T1 and A. When subjected to Western blot analysis with antibody to S1, both poly(A) RNA and isolated poly(A) tracts revealed bound S1 protein. The implications of these results for the possible interaction of poly(A) tracts of mRNA and the translational machinery of E coli are discussed.
Kalapos
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Boston Biomedical Research Institute, MA 02114, USA.
Paulus
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Sarkar
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D001426:Bacterial Proteins / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D001665:Binding Sites; D001667:Binding, Competitive; D015139:Blotting, Southern; D015153:Blotting, Western; D002352:Carrier Proteins / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D002845:Chromatography; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics; D015151:Immunoblotting; D011061:Poly A / Q000032:analysis / Q000378:metabolism*; D011132:Polyribosomes; D014176:Protein Biosynthesis; D012329:RNA, Bacterial / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D012269:Ribosomal Proteins / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism
D001426:Bacterial Proteins; D002352:Carrier Proteins; D012329:RNA, Bacterial; D012333:RNA, Messenger; D012269:Ribosomal Proteins; C015494:ribosomal protein S1; D011061:Poly A